During the past year, this laboratory has extended studies on mechanism of HIV fusion-entry, and on the development of novel antiviral proteins based on the entry mechanism to treat and prevent HIV infection. 1) Epitope masking within the HIV Env trimer. It is well known that several potential neutralizing epitopes on HIV Env are exposed on free gp120, but masked in the HIV trimer. The V1/V2 loops appear to contribute to masking. We have initiated experiments to test whether V1/V2 masking occurs within the same subunit or between subunits within the trimer. To this end, we have designed constructs with specific mutations and deletions; coexpression of different constructs will results in mixed trimers capable of fusion. The susceptibility patterns to neutralization by different monoclonal antibodies will enable us to determine whether masking of the corresponding epitopes occurs by intra- vs. inter-subunit mechanisms.2) Molecular mechanism of gp120-coreceptor interactions. Our previous studies on HIV neutralization by synthetic peptides derived from the extracellular regions of CCR5 have engendered ideas for designing soluble CCR5 mimics based on protein scaffolds. We have initiated studies with a new collaborator, Dr. W. Schief, U. Washington, who has extensive experience in this area, with particular respect to the HIV Env. 3) Potential role of neutralizing antibodies in selection for coreceptor usage during natural HIV infection. Based on our previous findings that a particular neutralizing epitope is preferentially exposed on CXCR4-using HIV-1 variants but masked on CCR5-specific variants, we will investigate whether antibodies which such specificities arise during natural infection and drive evolution of coreceptor usage. We will pursue collaborative studies of acute seroconverters with Dr. G. Shaw, U. Alabama and with SHIV-infected monkeys with W. Narayan, U. Kansas. 4) sCD4-17b, a novel anti-HIV neutralizing protein for potential use as a microbicide to prevent of HIV infection. Studies during the past year have focused on modifications of the protein with varying linker lengths between the sCD4 and 17b moieties. Our results indicate that molecules with sufficiently long linkers display extremely potent neutralization potency (IC50 5-20 nM) and breadth (against clades A, B, C, E; others to be tested); so far, every isolate examined has proven sensitive, in contrast to the best known broadly reactive monoclonal antibodies which all display comparatively limited breadth. We are also test variant linkers that give better yields of sCD4-17b expressed in Lactobacillus, toward the goal of producing a live microbicide that will colonize the vaginal tract and continuously secrete the neutralizing protein. 5) Immunotoxin to deplete HIV reservoirs. We have initiated studies with Dr. M. Martin to test the ability of 3B3-PE38 to deplete HIV infected cells from macaques infected with SHIV. Dr. Martin has extensive experience with testing antivirals in this system, and we are particularly interested in testing combinations of 3B3-PE38 with PMPA, an RT inhibitor. In addition, our collaborator Dr. Ira Pastan has initiated communications with a biotech company experienced in antibody-based therapeutics for possible collaboration to move 3B3-PE38 into Phase 1 clinical trials. We have received strong expressions of interest from several clinical investigators at extramural institutions (S. Deeks, UCSF; D. Margolis, UNC), as well as from the director of the ACTG (Alan Landay, Rush).